Characterization of Human Papillomavirus-11 mRNAs Expressed in the Context of Autonomously Replicating Viral Genomes

VIROLOGY

220, 177–185 (1996) 0298

ARTICLE NO.

Characterization of Human Papillomavirus-11 mRNAs Expressed in the Context of Autonomously Replicating Viral Genomes KAREN JANE RENAUD and LEX M. COWSERT1 Department of Infectious Diseases, Isis Pharmaceuticals, Inc., 2280 Faraday Avenue, Carlsbad, California 92008 Received February 6, 1996; accepted April 8, 1996 We have previously adapted an experimental system which supports autonomous replication of human papillomavirus11 (HPV-11) genomic DNA in a human squamous carcinoma cell line (SCC-4) following electroporation of linearized viral DNA. This system allows evaluation of HPV-11 transcription in the context of replicating viral genomes. RNA was isolated from cells following transfection, and first-strand cDNA was produced by reverse transcription using HPV-11-specific primers. The cDNAs were amplified using the polymerase chain reaction, and resulting DNA products were cloned and sequenced. Transcripts were identified that utilize the same splice donor and acceptor sites as transcripts characterized previously from human lesions. Three previously unreported splice junctions that employ novel combinations of those splice sites were also identified. We also detected these newly identified transcripts in laryngeal papillomas. A modified version of the 5* rapid amplification of cDNA ends method was used to identify transcriptional start sites of several of the HPV-11 transcripts. The family of mRNAs characterized in this replication system have the potential to encode all of the major HPV11 E-region proteins described to date. The data support the utility of this system as an experimental model for examining mechanisms of viral replication and transcription. q 1996 Academic Press, Inc.

INTRODUCTION

(Chow et al., 1987). Further studies employing PCR (Chiang et al., 1991; Rotenberg et al., 1989b), cDNA cloning (Nasseri et al., 1987), nuclease mapping (Smotkin et al., 1989; DiLorenzo and Steinberg, 1995), and retrovirusmediated gene transfer (Chiang et al., 1991; Rotenberg et al., 1989a) confirmed the structures of these transcripts and identified additional mRNAs, some of which utilize nonconsensus splice sites. Information from these studies has been useful in examining the role of various HPV-11 proteins in viral replication and transcription. For example, experiments employing the use of expression vectors to produce HPV11 proteins from heterologous promoters demonstrated that the E1 and E2 proteins were necessary and sufficient for DNA replication (Chiang et al., 1992). However, due to the lack of an in vitro system permissive to autonomous replication of HPV-11 DNA, these studies could not address the roles of HPV-11 gene products in the context of the replicating viral genome. Identification and accurate mapping of each mRNA species are crucial for understanding the roles of viral gene products in replication and transcription, as well as how the expression of these products is regulated. A thorough characterization of the transcripts expressed from autonomously replicating viral DNA is especially important because some of the viral gene products function as regulatory proteins in transcription and replication. Until recently, no culture system had been available in which to study HPV-11 DNA replication and transcrip-

Papillomaviruses are small, nonenveloped, DNA viruses that infect mammals in a species- and host cellspecific manner. Nearly 70 different genotypes of human papillomaviruses (HPVs) have been isolated (de Villiers, 1994). The genomic DNAs of several HPV types have been cloned and characterized. HPV genomes consist of a double-stranded, circular DNA of about 7900 bp, which can be divided into three regions: the long control region, containing cis-acting genetic elements; the early region (E-region), which contains genes expressed in nonproductively infected cells; and the late region (L-region), containing genes that are expressed only in productively infected cells (for reviews, see Chow and Broker, 1994; Howley, 1990; zur Hausen and de Villiers, 1994). One of the most-studied human papillomaviruses is HPV-11 (Dartmann et al., 1986) which is associated with condylomata acuminata (genital warts) and respiratory papillomatosis. Various groups have made use of the HPV-11 cDNA (Gissmann et al., 1982) to identify and characterize a number of HPV-11 transcripts. Electronmicroscopic R-loop studies of human condyloma mRNA, in conjunction with an examination of the HPV-11 sequence for the presence of consensus splice sites, provided the first descriptions of HPV-11 mRNA structure

1 To whom correspondence and reprint requests should be addressed. Fax: (619) 931-0209; E-mail: [email protected].

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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tion in the context of the viral genome. Del Vecchio et al. (1992) demonstrated that HPV-11 DNA can replicate autonomously when transfected into cultured SCC-4 cells. We have adapted this system to study the effects of antiviral compounds on HPV-11 DNA replication (P. R. Clark and L. M. Cowsert, manuscript submitted for publication). In order to further investigate the effects of these compounds on viral transcription, we needed to identify and characterize the mRNA species expressed in this system. Since the full productive functions of HPV-11, including transcription of L-region genes, are only expressed in terminally differentiated keratinocytes, this study focuses on E-region transcripts. We report the expression of HPV-11 transcripts encoding all of the major E-region proteins in SCC-4 cells containing autonomously replicating HPV-11 DNA. The nucleotide sequences spanning splice junctions of the transcripts were determined from cDNAs derived from viral mRNAs. Transcriptional start sites for some of the transcripts were mapped. Novel transcripts identified in our system were confirmed to be expressed in vivo. The results support the use of this system as a model for investigating HPV-11 E-region gene expression. MATERIALS AND METHODS

52.5 mM KCl, 1 mM MgCl2 , 200 mM dNTPs, 10 pmol of each HPV-11-specific primer, and 2 U AmpliTaq DNA polymerase (Perkin – Elmer Corporation, Norwalk, CT). Amplification of cDNA was performed in a Perkin – Elmer DNA thermal cycler as follows. Incubation was at 947 for 5 min; 35 cycles of denaturation for 1 min at 947, annealing for 30 sec at 577, elongation for 2 min at 727; and incubation at 727 for 10 min. Amplification products were visualized on a 3% agarose gel and isolated from gel slices by purification on glass powder (Vogelstein and Gillespie, 1979). In some cases, 10% of the isolated PCR product was reamplified using the same PCR primers, and the product(s) of the second amplification was gel purified for cloning. The cDNAs were ligated into Bluescript vector (Stratagene, La Jolla, CA) by utilizing restriction enzyme sites encoded in the linker sequences of the PCR primers. Plasmid DNA was prepared by the alkaline lysis method (Sambrook et al., 1989), purified on a silica membrane spin column (GlassMAX; Gibco-BRL), and sequenced using the Sequenase Version 2.0 DNA sequencing kit (Amersham Life Sciences, Arlington Heights, IL), according to the manufacturer’s instructions. Samples were separated on a 6% polyacrylamide gel containing 8 M urea. For each cDNA, the nucleotide sequence spanning the splice junction was determined, including at least 50 bp on each side of the junction site.

Transfections and RNA isolation Isolation of cDNAs by 5*-RACE Linearized full-length HPV-11 genomic DNA obtained by excision from the plasmid pUC19 (Yanisch-Perron et al., 1985) was transfected into human squamous carcinoma (SCC-4) cells by electroporation, as described by Del Vecchio et al. (1992). Total RNA was isolated from transfected cells by the guanidine isothiocyanate method (Chirgwin et al., 1979). RNA was suspended in RNasefree water and stored at 0807. Laryngeal papilloma biopsy specimens were a generous gift from Dr. Bettie Steinberg (Long Island Jewish Medical Center). RNA was prepared from sample homogenates by the same method used for transfected cells. Isolation of cDNAs by RT-PCR Ten micrograms of total RNA from HPV-11-electroporated SCC-4 cells was annealed with 2.5 pmol of an HPV-11-specific oligonucleotide (Operon Technologies, Alameda, CA; see Table 1 for sequences) at 707 for 10 min. Reverse transcription was carried out in a 20-ml reaction volume with 200 U Superscript II reverse transcriptase (Gibco-BRL Life Technologies, Gaithersburg, MD), 500 mM dNTPs, in 50 mM Tris – HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2 , and 10 mM DTT, at 427 for 1 hr. The reaction was stopped by incubation at 707 for 15 min. Five microliters was used directly for each amplification reaction in a total volume of 50 ml. The final reaction mixture contained 23 mM Tris – HCl, pH 8.4,

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To isolate cDNAs containing transcriptional start sites of HPV-11 transcripts, the 5*-RACE system for rapid amplification of cDNA ends (Gibco-BRL) was used, with modifications based on a procedure described by Ba¨hring et al. (1994). Briefly, 10 mg of total RNA was reverse transcribed using an HPV-11-specific primer according to the manufacturer’s instructions. The first-strand cDNA was treated with 2 U Escherichia coli RNase H for 10 min at 557 to remove bound template and purified on GlassMAX columns. Twenty to fifty percent of the firststrand cDNA was tailed with oligo(dG) by incubation with 15 U terminal deoxynucleotidyl transferase and 200 mM dGTP in 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2 for 10 min at 377. Twenty to forty-five percent of the dG-tailed cDNA was amplified by PCR using an oligo(dC) linker primer and an HPV-11-specific primer. The 50-ml reaction mixture contained 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 1 mM MgCl2 , 200 mM dNTPs, 10 pmol of each primer, and 2 U AmpliTaq DNA polymerase. The cycling protocol was performed as described for the isolation of cDNAs by RT-PCR. PCR products were isolated from agarose gels, cloned, and sequenced as described above. RT-PCR gel analysis Total RNA from HPV-11-transfected SCC-4 cells or laryngeal papilloma biopsy specimens was incubated in

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TABLE 1 Oligonucleotide Primers for Generation of cDNAs Speciesa i, vii

ii, iii

iv

v, vi

viii, ix

zA

zB

Primerb

Sequencec

Nucleotides

RT 3156 dC-linker DN 3109 RT 3800 UP 347 DN 3751 RT 3156 UP 1231 DN 3109 RT 3800 UP 1231 DN 3751 RT 3800 dC-linker DN 3751 RT 846 dC-linker DN 816 RT 585 dC-linker DN 551

5*-CATGAGTCGTTGTCCTGCAG-3* 5*-gcagatctcgagccccccccccccccc-3* 5*-cggaattcCATTACATTGTCTTCACAGC-3* 5*-CCACCTTATGCCTAATGGTG-3* 5*-ccatcgatTGCTGCATATGCACCTACAG-3* 5*-cggaattcCTGACGTTGTTCCTCACTGC-3* 5*-CATGAGTCGTTGTCCTGCAG-3* 5*-gcagatctGACAGTGGATATGGCTATTC-3* 5*-cggaattcCATTACATTGTCTTCACAGC-3* 5*-CCACCTTATGCCTAATGGTG-3* 5*-gcagatctGACAGTGGATATGGCTATTC-3* 5*-cggaattcCTGACGTTGTTCCTCACTGC-3* 5*-CCACCTTATGCCTAATGGTG-3* 5*-gcagatctcgagccccccccccccccc-3* 5*-cggaattcCTGACGTTGTTCCTCACTGC-3* 5*-TGAATCGTCCGCCATCCTTG-3* 5*-gcagatctcgagcccccccccccccc-3* 5*-cggaattcGGTGCGCAGATGGGACACAC-3* 5*-TCAGGAGGCTGCAGGTCTAG-3* 5*-gcagatctcgagccccccccccccccc-* 5*-cggaattcGGGTAACAAGTCTTCCATGC-3*

3156–3137 NAd 3109–3090 3800–3781 347–366 3751–3732 3156–3137 1231–1250 3109–3090 3800–3781 1231–1250 3751–3732 3800–3781 NA 3751–3732 846–827 NA 816–797 585–566 NA 551–532

a

Structure of mRNAs illustrated in Fig. 1. RT, primers used to produce first-strand cDNA; UP and DN, primers used in amplification step for cDNAs generated by RT-PCR; dC-linker and DN, primers used in amplification step for cDNAs generated by 5*-RACE. c Uppercase letters indicate HPV-11 sequences; lowercase letters indicate ‘‘linker’’ sequences that contain recognition sites for EcoRI or BglII restriction enzymes. d Not applicable. b

10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 10 mM MgCl2 , and either 0.2 units/ml DNase I (Boehringer Mannheim) or 10 ng/ml RNase (Boehringer Mannheim) at 377 for 1 hr. The samples were then subjected to reverse transcription and PCR amplification as described above for the isolation of cDNAs by RT-PCR. Samples from amplification reactions were electrophoresed in 2.5% agarose gels or 8% polyacrylamide gels and the DNA products were visualized by ethidium bromide staining. In some instances, where the amount of PCR product was not sufficient to be visualized by ethidium bromide staining, the products were analyzed by Southern blotting using an HPV-11 genomic DNA probe. Oligonucleotide primers used for RT-PCR were as follows, with ‘‘RT’’ designating a primer used for reverse transcription, ‘‘UP’’ designating an ‘‘upstream’’ PCR primer (complementary to the antisense strand of HPV-11), and ‘‘DN’’ designating a ‘‘downstream’’ PCR primer (complementary to the sense strand of HPV-11). RT 3156 (see Table 1); RT 3681, 5*-CAATGCCACGTTGAAGATGC-3* [nucleotides (nts) 3681–3662]; RT 3800 (see Table 1); UP 109, 5*-GTAAAGATGCCTCCACGTCT-3* (nts 109–128); UP 114, 5*-GATGCCTCCACGTCTGCAAC-3* (nts 114–133); UP 821, 5*-CCATAACAAGGATGGCGGAC-3* (nts 821–840); UP 1231 (see Table 1); UP 1381, 5*-GACAGCACCCGAGAGCATGC-3*; DN 2691, 5*-GCTGGACGACAGTCTTTCAA-3* (nts 2691–

To characterize the E-region HPV-11 mRNAs expressed in SCC-4 cells containing transiently replicating HPV-11 DNA, we used RT-PCR or 5*-RACE to generate HPV-11 cDNAs from transfected SCC-4 cell RNA. The RT-PCR and 5*-RACE products were ligated into a cloning vector and sequenced to determine the splice junctions contained in the transcripts from which the cDNAs were obtained. Sequence data from cDNAs obtained by 5*-RACE additionally provided information on transcriptional start sites. Figure 1 depicts the mRNA species identified using these procedures. Table 1 lists the primers used in the reverse transcription and amplification steps. Transcripts were identified that contain splice junctions utilizing all possible combinations of the splice donors at nt 847, nt 1272, and nt 1459 with the splice acceptors at nt 2622, nt 3325, and nt 3377. Each of these splice donor and acceptor sites has been described previously from in vivo or retrovirus-mediated gene transfer

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2672); DN 3109 (see Table 1); DN 3367, 5*-GGTGTATGTAGTAGGTTCAG-3* (nts 3367–3348); DN 3405, 5*-GTGCAGGCGGACACTGTAGG-3* (nts 3405–3386); and DN 3751 (see Table 1). RESULTS

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FIG. 1. Summary of HPV-11 mRNA structures. (Top) Open reading frames in the early region of the HPV-11 genome. (Bottom) Structures of mRNAs detected in the transfected SCC-4 cells. Roman numerals i–x are used to designate transcripts according to splice junction, with letter subscripts used to distinguish transcripts containing the same splice junction but with 5*-exons of different lengths. Each mRNA species is represented by a line, with splice junctions indicated by a break in the line and nucleotide locations of splice donor and acceptor sites shown. Transcriptional start sites are depicted as solid circles with nucleotide positions indicated. Dotted lines are drawn at the 5*-ends of transcripts for which transcriptional start sites were not determined, indicating the relative lengths of the 5*-exons as determined by RT-PCR agarose gel analysis. Solid portions of the lines represent regions encompassed in cDNAs and consequently sequenced. Dashed lines at the 3*-ends show putative structure beyond the downstream PCR primer, with the arrowhead located at the early poly(A) site, the presumed termination site for these transcripts. Coding potentials indicate the open reading frames contained in each transcript. (All transcripts also contain the E5a and E5b ORFs.) References refer to identification of similar transcripts by other methods: 1, Chiang et al., 1991; 2, Chow et al., 1987; 3, DiLorenzo and Steinberg, 1995; 4, Nasseri et al., 1987; 5, Rotenberg et al., 1989a; 6, Rotenberg et al., 1989b; 7, Smotkin et al., 1989; *, novel transcript (not previously identified).

studies (Chiang et al., 1991; Chow et al., 1987; Nasseri et al., 1987; Rotenberg et al., 1989a,b). However, not all combinations of these donors and acceptors had been reported before now. Splice junctions that have been previously described include 847Ú2622, 847Ú3325, 847Ú3377, 1272Ú3325, 1272Ú3377, and 1459Ú3325 (references indicated in Fig. 1). Transcripts containing splice junctions 1272Ú2622, 1459Ú2622, or 1459Ú3377 have not been previously identified. To verify that the novel transcripts are not an artifact of the transient replication system, we wanted to determine if these splice junction combinations are also utilized by HPV-11 transcripts in vivo. Figure 2 shows the results of gel analysis of RT-PCR products from transfected SCC-4 cells and laryngeal papillomas. Each

of the newly identified splice junction combinations from the transfected cells is also detected in the laryngeal papilloma, confirming that the novel mRNAs are transcribed in vivo. Because the PCR methods used in this study are not quantitative, no conclusions can be drawn regarding the relative levels of expression of the various transcripts. Several of the cDNAs from transfected SCC-4 cells were obtained by the modified 5*-RACE method described under Materials and Methods. The 5*-ends of these cDNAs were sequenced to identify the transcriptional start sites of the corresponding mRNAs (Fig. 3). These transcripts are depicted in Fig. 1 with solid circles at their 5*-ends. Sequence data for the 5*-RACE cDNAs are shown in

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FIG. 2. Analysis of transcript expression by RT-PCR. Total RNA was isolated from nontransfected (control) and transfected SCC-4 cells and a laryngeal papilloma specimen. The RNA samples were treated with DNase (D), RNase (R), or no enzyme (n), and subjected to RT-PCR as described under Materials and Methods. Transcript structures are depicted as described in Fig. 1. The nucleotide locations of oligonucleotide primers used for PCR amplification are indicated above the arrows. The lengths of the resulting PCR products include ‘‘linker’’ sequences contained in some of the primers. (A) Amplification products were separated on an agarose gel and analyzed by Southern blotting using an HPV-11 genomic probe. (B) Amplification products were separated on a polyacrylamide gel and visualized by ethidium bromide staining.

Fig. 3. The first several bands at the bottom of each gel image indicate sequence derived from the linker portion of the dC-linker primer. The stretch of consecutive Cs is derived from the portion of the dC-linker primer that

annealed to the oligo(dG) tail of the HPV-11 first-strand cDNA. Following this is a G that is found in neither the primer nor the HPV-11 sequence. This is followed by HPV-11 sequence. The G between the dC-linker and

FIG. 3. Identification of transcriptional start sites from cDNAs generated using a modified 5*-RACE method. cDNAs were cloned and sequenced as described under Materials and Methods. Gels are numbered according to the corresponding transcript illustrated in Fig. 1. Nucleotide positions of start sites are indicated. Alternate transcriptional start sites not shown here are as follows: for 1459Ú2622 splice, nts 1363, 1372, 1374, and 1416; for 1459Ú3325 splice, nts 1372, 1374, and 1378. Images of sequencing gels were scanned on a Molecular Dynamics Model 400E PhosphorImager using ImageQuant software.

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HPV-11 sequences is presumed to be derived from the 7-methylguanosine (m7G) cap of the RNA transcript, indicating that the first nucleotide of the HPV-11 sequence corresponds to a transcriptional start site. This method has previously been used to map other transcriptional start sites (Ba¨hring et al., 1994; Hirzmann et al., 1993). The 5*-RACE cDNA sequences reveal the presence of transcripts with the following structures: a transcriptional start site at nt 743 and a splice junction at 847Ú2622 (iA); a transcriptional start site at nt 1416 and a splice junction at 1459Ú2622 (viiA); a transcriptional start site at nt 1374 and a splice junction at 1459Ú3325 (viiiA); and a transcriptional start site at 1374 and a splice junction at 1459Ú3377 (ixA). No additional cDNAs containing the 847Ú2622 or the 1459Ú3377 splice junction were obtained by 5*-RACE. Multiple cDNAs were isolated that contained the other two splice junctions, and several of these had transcriptional start sites that varied slightly from those shown in Fig. 3. For example, we sequenced five cDNAs containing the 1459Ú3325 splice junction. Three of these had a transcriptional start site at nt 1374, one at nt 1372, and one at nt 1378. These start sites have not been previously identified and could indicate the presence of a novel promotor. Figure 3 (zA) provides data that may indicate a transcriptional start site at nt 445. However, the HPV-11 sequence contains a G at nt position 444. It follows that the G in the 5*-RACE cDNA sequence located after the string of Cs could be derived either from a m7G cap or from the G at nt 444 in the HPV-11 sequence. We repeated the isolation of cDNAs by 5*-RACE and sequenced a total of eight cDNAs that had 5*-ends identical to that shown in Fig. 3 (zA). Still, due to the presence of a G at nt 444 in the HPV-11 sequence, we cannot state conclusively that nt 445 is a transcriptional start site based on 5*-RACE cDNA sequence data alone. Other groups have identified HPV-11 transcripts initiating at the E7 promotor, with transcriptional start sites at around nt 260 (DiLorenzo and Steinberg, 1995; Smotkin et al., 1989). We are currently employing other experimental methods to confirm the presence of E7 transcripts with an alternate start site at nt 445. The data in Fig. 3 (zB) indicate that a mRNA species with a transcriptional start site at nt 94 is expressed in our system. This is in agreement with R-loop, RNase protection, and S1/ExoVII mapping studies (Chin et al., 1989; Chow et al., 1987; Smotkin et al., 1989) that identified a transcriptional start at nt 100, nt 99, and nt 90, respectively. Our cDNA contained only sequences from nt 94 to nt 551, due to the design of the downstream HPV-11-specific PCR primer, so the transcript from which it was derived could have contained any one of the splice junctions we identified. cDNAs isolated by the modified 5*-RACE method enabled us to map transcriptional start sites as described above. However, the 5*-ends of cDNAs obtained by the

RT-PCR method are defined by the upstream primers used in the amplification steps. To determine if transcripts contained additional 5*-sequences upstream of the PCR primer location, we used each of our upstream PCR primers with each downstream primer (see Materials and Methods) to generate RT-PCR products from transfected SCC-4 cell RNA. The presence or absence of amplification products from each combination, as well as the product sizes, was analyzed by gel electrophoresis to determine the most 5* sequences we could detect for each transcript. The results from these analyses are included in Fig. 1. The 3*-ends of all of the cDNAs we isolated are defined by the downstream primers used in the PCR reactions. We assume that the transcripts from which our cDNAs were derived extend to the early poly(A) site described in previous studies (Chow et al., 1987; Nasseri et al., 1987), located around nt 4400. Figure 1 shows the open reading frames (ORFs) contained in each transcript, assuming each has its 3*-end at nt 4400. As discussed below, the translation efficiency of an ORF in a multicistronic transcript depends upon its position relative to other ORFs. We would expect, then, that not all of the ORFs listed would be expressed from every transcript.

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DISCUSSION We have shown that alternatively processed E-region mRNAs are expressed in cultured SCC-4 cells autonomously replicating HPV-11 DNA. It is important to note that these transcripts were produced in the context of the HPV-11 viral genome alone, without cotransfection of regulatory protein expression vectors. Due to the very low abundance of many HPV-11 mRNAs expressed both in tissues and in the cultured SCC-4 cells, detection by Northern blot techniques is not practical. The long stretches of sequence shared by different transcripts further complicate attempts to distinguish between the mRNAs by methods such as Northern analysis and ribonuclease protection assay. We therefore characterized the HPV-11 transcripts using PCR amplification techniques. Although the use of HPV-11-specific primers for the generation of first-strand cDNA will necessarily bias the resulting products, our primer choice and use of multiple combinations of primers were designed to maximize the detection of any transcripts derived from the E-region of the viral genome. Indeed, using these methods in the SCC-4 transfection system, we detected transcripts that had not been previously identified by any of the methods utilized by other groups. Still, it is possible that additional E-region mRNA species exist that were not detected by our methods. HPV-11 transcripts were characterized by sequencing cDNAs obtained by RT-PCR or by a modified 5*-RACE method. Splice junctions were determined from cDNAs obtained by both methods, while use of the 5*-RACE

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method also enabled identification of transcriptional start sites. From these data, we have demonstrated that the transfected SCC-4 cells express every major E-region transcript previously found in human condylomas. Novel transcripts identified in our system were found to be expressed in laryngeal papillomas. Several groups have employed various methods to characterize HPV-11 mRNAs. Many of these studies characterized transcripts from human condylomata (Chiang et al., 1991; Chow et al., 1987; Nasseri et al., 1987; Rotenberg et al., 1989b; Smotkin et al., 1989) or laryngeal papillomas (DiLorenzo and Steinberg, 1995). Others used retrovirus-mediated gene transfer to identify possible splice junctions (Chiang et al., 1991; Rotenberg et al., 1989a). The results presented here indicate that the mRNAs expressed in transfected SCC-4 cells utilize the same splice donors and acceptors as had been identified from these previous studies. Our data demonstrate the presence of transcripts containing splice junctions that utilize all possible combinations of the splice donor sites at nts 847, 1272, and 1459 with the splice acceptor sites at nts 2622, 3325, and 3377. Six of these splice junctions have been previously identified while three have not. The novel transcripts were confirmed by RTPCR analysis to be expressed in laryngeal papillomas, demonstrating that these transcripts are not unique to the culture system used in this study. The family of mRNAs characterized in the transient replication system have the potential to encode all of the major HPV-11 E-region proteins described to date, with the exception of an E1MÚE2C fusion protein. Moreover, with the identification of new transcriptional start sites and splice junctions, we have discovered mRNAs that encode proteins such as E2Ca and E5a without the presence of other upstream ORFs. As discussed below, this set of transcripts encodes every E-region protein, with minimal redundancy and seldom requiring reinitiation of translation. The E1 and E2 proteins are necessary for viral replication (Chiang et al., 1992; Del Vecchio et al., 1992), so we would expect to find transcripts encoding these proteins in this system. Although we did not identify an E1-specific promoter, the full-length E1 protein could be expressed from a transcript with the E6 or E7 promoter. We did detect a transcript by RT-PCR (x) that includes nts 821– 3109 with no splice junction, thus containing the entire E1 ORF. Three other mRNAs encode N-terminal portions of the E1 ORF. Two short E1 proteins, E1M and E1Ma, and an E1ÚE4 fusion protein could be translated from species iv, viiB , and viiiB , respectively. The 1272Ú2622 splice junction contained in species iv has not been previously reported. By R-loop mapping of HPV-6 RNA preparations, Chow et al. (1987) identified a transcript with the 1272Ú3325 splice junction and a 5*-end near nt 700 that would also encode the E1M protein. However, they did not detect this transcript in HPV-11 RNA preparations.

The HPV-11 transcripts had 5*-ends located within the E1 ORF. Likewise, in our studies, none of the transcripts containing the 1272Ú3325 splice junction had 5*-sequences that included the E1 AUG. It appears, then, that the newly identified species iv may be the only HPV-11 transcript that can express E1M. Species vii is the first transcript identified that encodes the E1Ma protein. An E1MaÚE4 fusion protein could be translated from species viiiB . This transcript was identified by Chiang et al. (1991) as a cDNA obtained by retrovirus-mediated gene transfer. However, we did not detect a transcript encoding the E1MÚE2C fusion protein also described by Chiang et al. (1991). Species vi contains the 1272Ú3377 splice junction found in their cDNA but does not contain 5*-sequences extending beyond the start of the E1 ORF. Further studies to precisely map the transcriptional start sites of these transcripts will be necessary to resolve whether an E1MÚE2C transcript is expressed in the transfected cells in the context of the HPV-11 viral genome. We identified four transcripts in this system that could express the E2 transactivator. Two of these contain the 847Ú2622 splice junction, but with different-sized 5*-exons. Species iB encodes the E6 and E7 proteins in addition to E2. Based on studies of translation from papillomavirus mRNAs with multiple open reading frames (Barbosa and Wettstein, 1988a,b; Iftner et al., 1990), we would not expect the E7 protein to be translated from this mRNA. The shorter species, iA , would express only the E2 protein. These two mRNAs have each been found in human condylomas and by retrovirus-mediated gene transfer (Chow et al., 1987; Rotenberg et al., 1989a,b). Full-length E2 could also be translated from the novel species iv, assuming that reinitiation of translation occurs after translation of E1M. The relative positions of the E1M termination codon and the E2 AUG in this transcript should allow for efficient reinitiation of translation (Kozak, 1987; Peabody and Berg, 1986; Peabody et al., 1986). The fourth E2 mRNA (viiA) appears to be transcribed from a novel promoter and contains the novel splice junction 1459Ú2622. This transcript and species iA differ only in their approximately 100-bp 5*-exons, which contain untranslated sequences. It is intriguing to speculate that E2 is translated primarily from these two species (iA and viiA), and that the expression of the two transcripts is differentially regulated by their particular promoters. Two E2C repressor proteins have been described for bovine papillomavirus (Choe et al., 1989; Hubbert et al., 1988; Lambert et al., 1989, 1987), but until now, only one had been identified for HPV-11 (Rotenberg et al., 1989b). We have detected a transcript encoding E2C (species v), containing the same splice junction as previously identified by R-loop mapping and PCR cloning (Chow et al., 1987; Rotenberg et al., 1989b). Although none of these studies detected 5*-sequences that include the E1 AUG, the transcriptional start for this mRNA has not been pre-

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cisely mapped. Should the transcript indeed encode E1M, it would be unlikely that E2C would be expressed efficiently from this mRNA as the relative locations of the E1M and E2C ORFs would probably preclude reinitiation of translation (Kozak, 1987; Peabody and Berg, 1986; Peabody et al., 1986). We have characterized a novel E2C HPV-11 transcript, species ixA , that encodes a smaller Cterminal E2 protein (E2Ca) and lacks any other upstream ORFs. The transcript contains a 5* untranslated region of about 85 nt followed by an AUG located at the 3*end of the 5*-exon. The novel 1459Ú3377 splice junction brings this AUG in-frame with the E2 ORF. The translated protein would consist of the C-terminal 149 amino acids of E2 preceded by the initiator methionine. This portion of the E2 protein is sufficient for DNA binding (Chin et al., 1989, 1988). The detection of these two E2-encoding transcripts expressed from autonomously replicating HPV-11 genomic DNA is important. In light of the fact that we did not detect any transcripts encoding an E1MÚE2C protein, it appears that E2C and E2Ca would play a significant role in repression. The E6 and E7 proteins are encoded in several of the transcripts we detected in this system. Species iB , ii, and viiB could each express the E6 protein. As mentioned above, we would not expect the E7 protein to be translated from mRNAs that also contained the entire E6 ORF, as reinitiation of translation should not occur for E7 (Barbosa and Wettstein, 1988a,b). We identified a possible E7 transcriptional start site at nt 445 (species zA). mRNAs transcribed from this promoter would not contain the E6 ORF and consequently could express the E7 protein. Species iii might also be able to express the E7 protein, depending on the location of the 5*-end of the transcript. We detected 5*-sequences extending to nt 347, which is located within the E6 ORF. Further mapping studies are underway to confirm the location of the 5*-end of this transcript. Various E4 transcripts have been detected in HPV-11 condylomas (Chiang et al., 1991; Chow et al., 1987; Nasseri et al., 1987; Rotenberg et al., 1989b; Smotkin et al., 1989). We detected transcripts encoding the E1iÚE4 and E1MaÚE4 proteins and one that could express an E4C protein. Each of these transcripts has been described previously, but the expression of an E4C protein was not addressed. It is possible that E4C is one of the proteins detected by anti-E4 antibodies in HPV-11 infected tissues (Brown et al., 1994, 1991, 1988; Tomita et al., 1991). Due to the design of oligonucleotide primers used in this study, none of our cDNAs contained sequences downstream of the E2 ORF. We assume that the transcripts terminate at the early poly(A) site, thus including the E5a and E5b ORFs in all of the mRNAs characterized here. Expression of these proteins from specific transcripts would depend on the efficiency of reinitiation of translation. Only the previously unreported species viiiA contains no ORFs upstream of E5a and E5b.

The novel promoter identified for the E5a/E5b transcript is also used by two other mRNAs discussed above. Species viiA encodes full-length E2 with no upstream ORFs, but uses a different promoter from the species iA E2 mRNA. Species ixA encodes a novel putative E2 repressor protein, E2Ca, with no upstream ORFs. Species viiiA encodes E5a and E5b with no upstream ORFs. Each of these mRNAs appears to be an important member of the set of early region transcripts expressed from the autonomously replicating HPV-11 genome. In this study we did not investigate the relative abundance of different transcripts. We cannot assume that transcription in the transfection system exactly mimics viral transcription in vivo. Indeed, HPV-11 mRNA levels are not the same even between lesions taken from different patients. Thus, the present lack of information regarding transcript levels does not invalidate this transcription system as a useful model for investigating HPV-11 transcription. Rather, we have laid the foundation for further investigation of viral mRNA expression, by identifying three novel splice junctions and a potential novel promoter. This information is crucial for the proper design of molecular probes to assay levels of mRNA expression. The results presented here confirm the expression of viral mRNA in SCC-4 cells containing autonomously replicating HPV-11 DNA. The early region transcripts characterized here utilize the same splice donor and acceptor sites as transcripts identified in vivo. Examination of the coding potentials of the set of transcripts characterized here indicates that all of the major early region proteins could be expressed from these mRNAs. These results support the use of this system as a valuable tool for examining HPV-11 viral transcription.

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ACKNOWLEDGMENTS We thank Paul R. Clark for expert advice and assistance with the culture and electroporation of SCC-4 cells, and Dr. Bettie M. Steinberg for the gift of laryngeal papilloma biopsy specimens. This work was supported by Small Business Initiative Research Grant CA52391-03 from the National Cancer Institute.

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